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Abstract:

The present invention relates to isolated polypeptides having
antimicrobial activity and isolated polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid constructs,
vectors, and host cells comprising the polynucleotides as well as methods
for producing and using the polypeptides.

Claims:

1-56. (canceled)

57. A method for killing or inhibiting growth of microbial cells
comprising contacting the microbial cells with an antimicrobial
polypeptide, wherein the antimicrobial peptide is encoded by a
polynucleotide which hybridizes under high stringency conditions with (i)
nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA sequence contained
in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) the complementary strand
of (i) or (ii), wherein the high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3%
SDS, 200 mg/ml sheared and denatured salmon sperm DNA, and 50% formamide,
following standard Southern blotting procedures for 12 to 24 hours,
followed by washing three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 65.degree. C.

58. The method of claim 57, wherein the antimicrobial peptide is encoded
by a polynucleotide which hybridizes under very high stringency
conditions with (i) nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA
sequence contained in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) the
complementary strand of (i) or (ii), wherein the very high stringency
conditions are defined as prehybridization and hybridization at
42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and
denatured salmon sperm DNA, and 50% formamide, following standard
Southern blotting procedures for 12 to 24 hours, followed by washing
three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 70.degree.
C.

59. A method for killing or inhibiting growth of microbial cells
comprising contacting the microbial cells with an antimicrobial
polypeptide, wherein the antimicrobial peptide has at least 90% identity
with amino acids 1 to 42 of SEQ ID NO:2.

60. The method of claim 59, wherein the antimicrobial peptide has at
least 95% identity with amino acids 1 to 42 of SEQ ID NO:2.

61. The method of claim 59, wherein the antimicrobial peptide has at
least 96% identity with amino acids 1 to 42 of SEQ ID NO:2.

62. The method of claim 59, wherein the antimicrobial peptide has at
least 97% identity with amino acids 1 to 42 of SEQ ID NO:2.

63. The method of claim 59, wherein the antimicrobial peptide has at
least 98% identity with amino acids 1 to 42 of SEQ ID NO:2.

64. The method of claim 59, wherein the antimicrobial peptide has at
least 99% identity with amino acids 1 to 42 of SEQ ID NO:2.

[0002] The present invention relates to isolated polypeptides having
antimicrobial activity and isolated polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid constructs,
vectors, and host cells comprising the polynucleotides as well as methods
for producing and using the polypeptides.

BACKGROUND OF THE INVENTION

[0003] It is an object of the present invention to provide polypeptides
having antimicrobial activity and polynucleotides encoding the
polypeptides.

SUMMARY OF THE INVENTION

[0004] The present invention relates to isolated polypeptides having
antimicrobial activity selected from the group consisting of: [0005]
(a) a polypeptide having an amino acid sequence which has at least 60%
identity with amino acids 1 to 42 of SEQ ID NO:2; [0006] (b) a
polypeptide which is encoded by a nucleotide sequence which hybridizes
under at least medium stringency conditions with (i) nucleotides 145 to
270 of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides 1 to
270 of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii); and
[0007] (c) a variant comprising a conservative substitution, deletion,
and/or insertion of one or more amino acids of amino acids 1 to 42 of SEQ
ID NO:2.

[0008] The present invention also relates to isolated polynucleotides
encoding polypeptides having antimicrobial activity, selected from the
group consisting of: [0009] (a) a polynucleotide encoding a polypeptide
having an amino acid sequence which has at least 60% identity with amino
acids 1 to 42 of SEQ ID NO:2; [0010] (b) a polynucleotide having at least
60% identity with nucleotides 145 to 270 of SEQ ID NO:1, and [0011] (c) a
polynucleotide which hybridizes under at least medium stringency
conditions with (i) nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA
sequence contained in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) a
complementary strand of (i) or (ii).

[0013] The present invention also relates to methods for producing such
polypeptides having antimicrobial activity comprising (a) cultivating a
recombinant host cell comprising a nucleic acid construct comprising a
polynucleotide encoding the polypeptide under conditions conducive for
production of the polypeptide; and (b) recovering the polypeptide.

[0014] The present invention also relates to methods of using the
polypeptides and polynucleotides of the invention.

DEFINITIONS

[0015] Antimicrobial activity: The term "antimicrobial activity" is
defined herein as an activity which is capable of killing or inhibiting
growth of microbial cells. In the context of the present invention the
term "antimicrobial" is intended to mean that there is a bactericidal
and/or a bacteriostatic and/or fungicidal and/or fungistatic effect
and/or a virucidal effect, wherein the term "bactericidal" is to be
understood as capable of killing bacterial cells. The term
"bacteriostatic" is to be understood as capable of inhibiting bacterial
growth, i.e. inhibiting growing bacterial cells. The term "fungicidal" is
to be understood as capable of killing fungal cells. The term
"fungistatic" is to be understood as capable of inhibiting fungal growth,
i.e. inhibiting growing fungal cells. The term "virucidal" is to be
understood as capable of inactivating virus. The term "microbial cells"
denotes bacterial or fungal cells (including yeasts).

[0016] In the context of the present invention the term "inhibiting growth
of microbial cells" is intended to mean that the cells are in the
non-growing state, i.e., that they are not able to propagate.

[0017] For purposes of the present invention, antimicrobial activity may
be determined according to the procedure described by Lehrer et al.,
Journal of Immunological Methods, Vol. 137 (2) pp. 167-174 (1991).
Alternatively, antimicrobial activity may be determined according to the
NCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute;
formerly known as National Committee for Clinical and Laboratory
Standards).

[0018] Polypeptides having antimicrobial activity may be capable of
reducing the number of living cells of Escherichia coli (DSM 1576) to
1/100 after 8 hours (preferably after 4 hours, more preferably after 2
hours, most preferably after 1 hour, and in particular after 30 minutes)
incubation at 20° C. in an aqueous solution of 25% (w/w);
preferably in an aqueous solution of 10% (w/w); more preferably in an
aqueous solution of 5% (w/w); even more preferably in an aqueous solution
of 1% (w/w); most preferably in an aqueous solution of 0.5% (w/w); and in
particular in an aqueous solution of 0.1% (w/w) of the polypeptides
having antimicrobial activity.

[0019] Polypeptides having antimicrobial activity may also be capable of
inhibiting the outgrowth of Escherichia coli (DSM 1576) for 24 hours at
25° C. in a microbial growth substrate, when added in a
concentration of 1000 ppm; preferably when added in a concentration of
500 ppm; more preferably when added in a concentration of 250 ppm; even
more preferably when added in a concentration of 100 ppm; most preferably
when added in a concentration of 50 ppm; and in particular when added in
a concentration of 25 ppm.

[0020] Polypeptides having antimicrobial activity may be capable of
reducing the number of living cells of Bacillus subtilis (ATCC 6633) to
1/100 after 8 hours (preferably after 4 hours, more preferably after 2
hours, most preferably after 1 hour, and in particular after 30 minutes)
incubation at 20° C. in an aqueous solution of 25% (w/w);
preferably in an aqueous solution of 10% (w/w); more preferably in an
aqueous solution of 5% (w/w); even more preferably in an aqueous solution
of 1% (w/w); most preferably in an aqueous solution of 0.5% (w/w); and in
particular in an aqueous solution of 0.1% (w/w) of the polypeptides
having antimicrobial activity.

[0021] Polypeptides having antimicrobial activity may also be capable of
inhibiting the outgrowth of Bacillus subtilis (ATCC 6633) for 24 hours at
25° C. in a microbial growth substrate, when added in a
concentration of 1000 ppm; preferably when added in a concentration of
500 ppm; more preferably when added in a concentration of 250 ppm; even
more preferably when added in a concentration of 100 ppm; most preferably
when added in a concentration of 50 ppm; and in particular when added in
a concentration of 25 ppm.

[0022] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more preferably at
least 60%, more preferably at least 70%, more preferably at least 80%,
even more preferably at least 90%, most preferably at least 95%, and even
most preferably at least 100% of the antimicrobial activity of the
polypeptide consisting of the amino acid sequence shown as amino acids 1
to 42 of SEQ ID NO:2.

[0023] Defensin: The term "defensin" as used herein refers to polypeptides
recognized by a person skilled in the art as belonging to the defensin
class of antimicrobial peptides. To determine if a polypeptide is a
defensin according to the invention, the amino acid sequence is
preferably compared with the hidden markov model profiles (HMM profiles)
of the PFAM database by using the freely available HMMER software package
(see Example 6).

[0025] The defensins may belong to the alpha-defensin class, the
beta-defensin class, the theta-defensin class, the insect (arthropod)
defensin class, the plant defensin class, the mussel defensin class, or
other defensin classes wherein the amino acid sequence comprises 6 or 8
cysteines and are structurally similar to any of the before-mentioned
defensin classes. The defensins may also be synthetic defensins sharing
the characteristic features of any of the defensin classes.

[0027] Isolated polypeptide: The term "isolated polypeptide" as used
herein refers to a polypeptide which is at least 20% pure, preferably at
least 40% pure, more preferably at least 60% pure, even more preferably
at least 80% pure, most preferably at least 90% pure, and even most
preferably at least 95% pure, as determined by SDS-PAGE.

[0028] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation which contains at
most 10%, preferably at most 8%, more preferably at most 6%, more
preferably at most 5%, more preferably at most 4%, at most 3%, even more
preferably at most 2%, most preferably at most 1%, and even most
preferably at most 0.5% by weight of other polypeptide material with
which it is natively associated. It is, therefore, preferred that the
substantially pure polypeptide is at least 92% pure, preferably at least
94% pure, more preferably at least 95% pure, more preferably at least 96%
pure, more preferably at least 96% pure, more preferably at least 97%
pure, more preferably at least 98% pure, even more preferably at least
99%, most preferably at least 99.5% pure, and even most preferably 100%
pure by weight of the total polypeptide material present in the
preparation.

[0029] The polypeptides of the present invention are preferably in a
substantially pure form. In particular, it is preferred that the
polypeptides are in "essentially pure form", i.e., that the polypeptide
preparation is essentially free of other polypeptide material with which
it is natively associated. This can be accomplished, for example, by
preparing the polypeptide by means of well-known recombinant methods or
by classical purification methods.

[0030] Herein, the term "substantially pure polypeptide" is synonymous
with the terms "isolated polypeptide" and "polypeptide in isolated form."

[0031] Identity: The relatedness between two amino acid sequences or
between two nucleotide sequences is described by the parameter
"identity".

[0032] For purposes of the present invention, the degree of identity
between two amino acid sequences may be determined by using the program
FASTA included in version 2.0× of the FASTA program package (see W.
R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological
Sequence Analysis", PNAS 85:2444-2448; and W. R. Pearson (1990) "Rapid
and Sensitive Sequence Comparison with FASTP and FASTA", Methods in
Enzymology 183:63-98). The scoring matrix used was BLOSUM50, gap penalty
was -12, and gap extension penalty was -2.

[0033] The degree of identity between two nucleotide sequences is
determined using the same algorithm and software package as described
above. The scoring matrix used was the identity matrix, gap penalty was
-16, and gap extension penalty was -4.

[0034] Alternatively, an alignment of two amino acid sequences is
determined by using the Needle program from the EMBOSS package
(http://emboss.org) version 2.8.0. The Needle program implements the
global alignment algorithm described in Needleman, S. B. and Wunsch, C.
D. (1970) J. Mol. Biol. 48, 443-453. The substitution matrix used is
BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.

[0035] The degree of identity between an amino acid sequence of the
present invention ("invention sequence"; e.g. amino acids 1 to 42 of SEQ
ID NO:2) and a different amino acid sequence ("foreign sequence") is
calculated as the number of exact matches in an alignment of the two
sequences, divided by the length of the "invention sequence" or the
length of the "foreign sequence", whichever is the shortest. The result
is expressed in percent identity.

[0036] An exact match occurs when the "invention sequence" and the
"foreign sequence" have identical amino acid residues in the same
positions of the overlap (in the alignment example below this is
represented by "|"). The length of a sequence is the number of amino acid
residues in the sequence (e.g. the length of amino acids 1 to 42 of SEQ
ID NO:2 is 42).

[0037] In the alignment example below, the overlap is the amino acid
sequence "HTWGER-NL" of Sequence 1; or the amino acid sequence
"HGWGEDANL" of Sequence 2. In the example a gap is indicated by a "-".

Alignment Example

##STR00001##

[0039] Polypeptide Fragment: The term "polypeptide fragment" is defined
herein as a polypeptide having one or more amino acids deleted from the
amino and/or carboxyl terminus of SEQ ID NO:2 or a homologous sequence
thereof, wherein the fragment has antimicrobial activity. Preferably a
polypeptide fragment of the invention retains all cysteine residues and
the amino acid residues between the cysteine residues.

Subsequence: The term "subsequence" is defined herein as a nucleotide
sequence having one or more nucleotides deleted from the 5' and/or 3' end
of SEQ ID NO:1 or a homologous sequence thereof, wherein the subsequence
encodes a polypeptide fragment having antimicrobial activity.

[0040] Allelic variant: The term "allelic variant" denotes herein any of
two or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be silent
(no change in the encoded polypeptide) or may encode polypeptides having
altered amino acid sequences. An allelic variant of a polypeptide is a
polypeptide encoded by an allelic variant of a gene.

[0041] Substantially pure polynucleotide: The term "substantially pure
polynucleotide" as used herein refers to a polynucleotide preparation
free of other extraneous or unwanted nucleotides and in a form suitable
for use within genetically engineered protein production systems. Thus, a
substantially pure polynucleotide contains at most 10%, preferably at
most 8%, more preferably at most 6%, more preferably at most 5%, more
preferably at most 4%, more preferably at most 3%, even more preferably
at most 2%, most preferably at most 1%, and even most preferably at most
0.5% by weight of other polynucleotide material with which it is natively
associated. A substantially pure polynucleotide may, however, include
naturally occurring 5' and 3' untranslated regions, such as promoters and
terminators. It is preferred that the substantially pure polynucleotide
is at least 90% pure, preferably at least 92% pure, more preferably at
least 94% pure, more preferably at least 95% pure, more preferably at
least 96% pure, more preferably at least 97% pure, even more preferably
at least 98% pure, most preferably at least 99%, and even most preferably
at least 99.5% pure by weight. The polynucleotides of the present
invention are preferably in a substantially pure form. In particular, it
is preferred that the polynucleotides disclosed herein are in
"essentially pure form", i.e., that the polynucleotide preparation is
essentially free of other polynucleotide material with which it is
natively associated. Herein, the term "substantially pure polynucleotide"
is synonymous with the terms "isolated polynucleotide" and
"polynucleotide in isolated form." The polynucleotides may be of genomic,
cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

[0042] cDNA: The term "cDNA" is defined herein as a DNA molecule which can
be prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic cell. cDNA lacks intron sequences
that are usually present in the corresponding genomic DNA. The initial,
primary RNA transcript is a precursor to mRNA which is processed through
a series of steps before appearing as mature spliced mRNA. These steps
include the removal of intron sequences by a process called splicing.
cDNA derived from mRNA lacks, therefore, any intron sequences.

[0043] Nucleic acid construct: The term "nucleic acid construct" as used
herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene or
which is modified to contain segments of nucleic acids in a manner that
would not otherwise exist in nature. The term nucleic acid construct is
synonymous with the term "expression cassette" when the nucleic acid
construct contains the control sequences required for expression of a
coding sequence of the present invention.

[0044] Control sequence: The term "control sequences" is defined herein to
include all components, which are necessary or advantageous for the
expression of a polynucleotide encoding a polypeptide of the present
invention. Each control sequence may be native or foreign to the
nucleotide sequence encoding the polypeptide. Such control sequences
include, but are not limited to, a leader, polyadenylation sequence,
propeptide sequence, promoter, signal peptide sequence, and transcription
terminator. At a minimum, the control sequences include a promoter, and
transcriptional and translational stop signals. The control sequences may
be provided with linkers for the purpose of introducing specific
restriction sites facilitating ligation of the control sequences with the
coding region of the nucleotide sequence encoding a polypeptide.

[0045] Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an appropriate
position relative to the coding sequence of the polynucleotide sequence
such that the control sequence directs the expression of the coding
sequence of a polypeptide.

[0046] Coding sequence: When used herein the term "coding sequence" means
a nucleotide sequence, which directly specifies the amino acid sequence
of its protein product. The boundaries of the coding sequence are
generally determined by an open reading frame, which usually begins with
the ATG start codon or alternative start codons such as GTG and TTG. The
coding sequence may a DNA, cDNA, or recombinant nucleotide sequence.

[0047] Expression: The term "expression" includes any step involved in the
production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.

Expression vector: The term "expression vector" is defined herein as a
linear or circular DNA molecule that comprises a polynucleotide encoding
a polypeptide of the invention, and which is operably linked to
additional nucleotides that provide for its expression.

[0048] Host cell: The term "host cell", as used herein, includes any cell
type which is susceptible to transformation, transfection, transduction,
and the like with a nucleic acid construct comprising a polynucleotide of
the present invention.

[0049] Modification: The term "modification" means herein any chemical
modification of the polypeptide consisting of the amino acids 1 to 42 of
SEQ ID NO:2 as well as genetic manipulation of the DNA encoding that
polypeptide. The modification(s) can be substitution(s), deletion(s)
and/or insertions(s) of the amino acid(s) as well as replacement(s) of
amino acid side chain(s); or use of unnatural amino acids with similar
characteristics in the amino acid sequence. In particular the
modification(s) can be amidations, such as amidation of the C-terminus.

[0050] Artificial variant: When used herein, the term "artificial variant"
means a polypeptide having antimicrobial activity produced by an organism
expressing a modified nucleotide sequence of SEQ ID NaI. The modified
nucleotide sequence is obtained through human intervention by
modification of the nucleotide sequence disclosed in SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Antimicrobial Activity

[0051] In a first aspect, the present invention relates to isolated
polypeptides having an amino acid sequence which has a degree of identity
to amino acids 1 to 42 of SEQ ID NO:2 (i.e., the mature polypeptide) of
at least 60%, preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, more preferably at
least 85%, even more preferably at least 90%, most preferably at least
95%, and even most preferably at least 97%, which have antimicrobial
activity (hereinafter "homologous polypeptides"). In a preferred aspect,
the homologous polypeptides have an amino acid sequence which differs by
ten amino acids, preferably by five amino acids, more preferably by four
amino acids, even more preferably by three amino acids, most preferably
by two amino acids, and even most preferably by one amino acid from amino
acids 1 to 42 of SEQ ID NO:2.

[0052] A polypeptide of the present invention preferably comprises the
amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; or a
fragment thereof that has antimicrobial activity. In a preferred aspect,
a polypeptide comprises the amino acid sequence of SEQ ID NO:2. In
another preferred aspect, a polypeptide comprises amino acids 1 to 42 of
SEQ ID NO:2, or an allelic variant thereof; or a fragment thereof that
has antimicrobial activity. In another preferred aspect, a polypeptide
comprises amino acids 1 to 42 of SEQ ID NO:2. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2
or an allelic variant thereof; or a fragment thereof that has
antimicrobial activity. In another preferred aspect, a polypeptide
consists of the amino acid sequence of SEQ ID NO:2. In another preferred
aspect, a polypeptide consists of amino acids 1 to 42 of SEQ ID NO:2 or
an allelic variant thereof; or a fragment thereof that has antimicrobial
activity. In another preferred aspect, a polypeptide consists of amino
acids 1 to 42 of SEQ ID NO:2.

[0053] In a second aspect, the present invention relates to isolated
polypeptides having antimicrobial activity which are encoded by
polynucleotides which hybridize under very low stringency conditions,
preferably low stringency conditions, more preferably medium stringency
conditions, more preferably medium-high stringency conditions, even more
preferably high stringency conditions, and most preferably very high
stringency conditions with (i) nucleotides 145 to 270 of SEQ ID NO:1,
(ii) the cDNA sequence contained in nucleotides 1 to 270 of SEQ ID NO:1,
(iii) a subsequence of (i) or (ii), or (iv) a complementary strand of
(i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,
Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,
N.Y.). A subsequence of SEQ ID NO:1 contains at least 100 contiguous
nucleotides or preferably at least 200 continguous nucleotides. Moreover,
the subsequence may encode a polypeptide fragment which has antimicrobial
activity.

[0054] The nucleotide sequence of SEQ ID NO:1 or a subsequence thereof, as
well as the amino acid sequence of SEQ ID NO:2 or a fragment thereof, may
be used to design a nucleic acid probe to identify and clone DNA encoding
polypeptides having antimicrobial activity from strains of different
genera or species according to methods well known in the art. In
particular, such probes can be used for hybridization with the genomic or
cDNA of the genus or species of interest, following standard Southern
blotting procedures, in order to identify and isolate the corresponding
gene therein. Such probes can be considerably shorter than the entire
sequence, but should be at least 14, preferably at least 25, more
preferably at least 35, and most preferably at least 70 nucleotides in
length. It is, however, preferred that the nucleic acid probe is at least
100 nucleotides in length. For example, the nucleic acid probe may be at
least 200 nucleotides, preferably at least 270 nucleotides. Both DNA and
RNA probes can be used. The probes are typically labeled for detecting
the corresponding gene (for example, with 32P, 3H, 35S,
biotin, or avidin). Such probes are encompassed by the present invention.

[0055] A genomic DNA or cDNA library prepared from such other organisms
may, therefore, be screened for DNA which hybridizes with the probes
described above and which encodes a polypeptide having antimicrobial
activity. Genomic or other DNA from such other organisms may be separated
by agarose or polyacrylamide gel electrophoresis, or other separation
techniques. DNA from the libraries or the separated DNA may be
transferred to and immobilized on nitrocellulose or other suitable
carrier material. In order to identify a clone or DNA which is homologous
with SEQ ID NO:1 or a subsequence thereof, the carrier material is used
in a Southern blot.

[0056] For purposes of the present invention, hybridization indicates that
the nucleotide sequence hybridizes to a labeled nucleic acid probe
corresponding to the nucleotide sequence shown in SEQ ID NO:1, its
complementary strand, or a subsequence thereof, under very low to very
high stringency conditions. Molecules to which the nucleic acid probe
hybridizes under these conditions can be detected using X-ray film.

[0057] In a preferred aspect, the nucleic acid probe is a polynucleotide
sequence which encodes the polypeptide of SEQ ID NO:2, or a subsequence
thereof. In another preferred aspect, the nucleic acid probe is SEQ ID
NO:1. In another preferred aspect, the nucleic acid probe is the mature
polypeptide coding region of SEQ ID NO:1.

[0058] For long probes of at least 100 nucleotides in length, very low to
very high stringency conditions are defined as prehybridization and
hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml
sheared and denatured salmon sperm DNA, and either 25% formamide for very
low and low stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally.

[0059] For long probes of at least 100 nucleotides in length, the carrier
material is finally washed three times each for 15 minutes using
2×SSC, 0.2% SDS preferably at least at 45° C. (very low
stringency), more preferably at least at 50° C. (low stringency),
more preferably at least at 55° C. (medium stringency), more
preferably at least at 60° C. (medium-high stringency), even more
preferably at least at 65° C. (high stringency), and most
preferably at least at 70° C. (very high stringency).

[0060] For short probes which are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at about
5° C. to about 10° C. below the calculated Tm using
the calculation according to Bolton and McCarthy (1962, Proceedings of
the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M
Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1
mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and
0.2 mg of yeast RNA per ml following standard Southern blotting
procedures.

[0061] For short probes which are about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in 6×SCC
plus 0.1% SDS for 15 minutes and twice each for 15 minutes using
6×SSC at 5° C. to 10° C. below the calculated
Tm.

[0062] In a third aspect, the present invention relates to artificial
variants comprising a conservative substitution, deletion, and/or
insertion of one or more amino acids of SEQ ID NO:2 or the mature
polypeptide thereof. Preferably, amino acid changes are of a minor
nature, that is conservative amino acid substitutions or insertions that
do not significantly affect the folding and/or activity of the protein;
small deletions, typically of one to about 30 amino acids; small amino-
or carboxyl-terminal extensions, such as an amino-terminal methionine
residue; a small linker peptide of up to about 20-25 residues; or a small
extension that facilitates purification by changing net charge or another
function, such as a poly-histidine tract, an antigenic epitope or a
binding domain.

[0064] In addition to the 20 standard amino acids, non-standard amino
acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric
acid, isovaline, and alpha-methyl serine) may be substituted for amino
acid residues of a wild-type polypeptide. A limited number of
non-conservative amino acids, amino acids that are not encoded by the
genetic code, and unnatural amino acids may be substituted for amino acid
residues. "Unnatural amino acids" have been modified after protein
synthesis, and/or have a chemical structure in their side chain(s)
different from that of the standard amino acids. Unnatural amino acids
can be chemically synthesized, and preferably, are commercially
available, and include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0065] Alternatively, the amino acid changes are of such a nature that the
physico-chemical properties of the polypeptides are altered. For example,
amino acid changes may improve the thermal stability of the polypeptide,
alter the substrate specificity, change the pH optimum, and the like.

[0066] Essential amino acids in the parent polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,
Science 244: 1081-1085). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity (i.e.,
antimicrobial activity) to identify amino acid residues that are critical
to the activity of the molecule. See also, Hilton of al., 1996, J. Biol.
Chem. 271: 4699-4708. The active site of the enzyme or other biological
interaction can also be determined by physical analysis of structure, as
determined by such techniques as nuclear magnetic resonance,
crystallography, electron diffraction, or photoaffinity labeling, in
conjunction with mutation of putative contact site amino acids. See, for
example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,
J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64.
The identities of essential amino acids can also be inferred from
analysis of identities with polypeptides which are related to a
polypeptide according to the invention.

[0067] Single or multiple amino acid substitutions can be made and tested
using known methods of mutagenesis, recombination, and/or shuffling,
followed by a relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,
1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO
95/22625. Other methods that can be used include error-prone PCR, phage
display (e.g., Lowman of al., 1991, Biochem. 30:10832-10837; U.S. Pat.
No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire
et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0068] Mutagenesis/shuffling methods can be combined with high-throughput,
automated screening methods to detect activity of cloned, mutagenized
polypeptides expressed by host cells. Mutagenized DNA molecules that
encode active polypeptides can be recovered from the host cells and
rapidly sequenced using standard methods in the art. These methods allow
the rapid determination of the importance of individual amino acid
residues in a polypeptide of interest, and can be applied to polypeptides
of unknown structure.

[0069] The total number of amino acid substitutions, deletions and/or
insertions of amino adds 1 to 42 of SEQ ID NO:2 is 10, preferably 9, more
preferably 8, more preferably 7, more preferably at most 6, more
preferably at most 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.

[0070] In a preferred embodiment, the polypeptides of the invention are
defensin polypeptides. In another embodiment, the polypeptides of the
invention comprise three di-cysteine bonds.

N-Terminal Extension

[0071] An N-terminal extension of the polypeptides of the invention may
suitably consist of from 1 to 50 amino acids, preferably 2-20 amino
acids, especially 3-15 amino acids. In one embodiment N-terminal peptide
extension does not contain an Arg (R). In another embodiment the
N-terminal extension comprises a kex2 or kex2-like cleavage site as will
be defined further below. In a preferred embodiment the N-terminal
extension is a peptide, comprising at least two Glu (E) and/or Asp (D)
amino acid residues, such as an N-terminal extension comprising one of
the following sequences: EAE, EE, DE and DD.

[0073] Insertion of a kex2 site or a kex2-like site have in certain cases
been shown to improve correct endopeptidase processing at the pro-peptide
cleavage site resulting in increased protein secretion levels.

[0074] In the context of the invention insertion of a kex2 or kex2-like
site result in the possibility to obtain cleavage at a certain position
in the N-terminal extension resulting in an antimicrobial polypeptide
being extended in comparison to the mature polypeptide shown as amino
acids 1 to 42 of SEQ ID NO:2.

Fused Polypeptides

[0075] The polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide of the invention or a fragment thereof. A fused polypeptide
is produced by fusing a nucleotide sequence (or a portion thereof)
encoding another polypeptide to a nucleotide sequence (or a portion
thereof) of the present invention. Techniques for producing fusion
polypeptides are known in the art, and include ligating the coding
sequences encoding the polypeptides so that they are in frame and that
expression of the fused polypeptide is under control of the same
promoter(s) and terminator.

Sources of Polypeptides Having Antimicrobial Activity

[0076] A polypeptide of the present invention may be obtained from
microorganisms of any genus. For purposes of the present invention, the
term "obtained from" as used herein in connection with a given source
shall mean that the polypeptide encoded by a nucleotide sequence is
produced by the source or by a strain in which the nucleotide sequence
from the source has been inserted. In a preferred aspect, the polypeptide
obtained from a given source is secreted extracellularly.

[0077] A polypeptide of the present invention may be a bacterial
polypeptide. For example, the polypeptide may be a gram positive
bacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus
circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide; or a Streptomyces
polypeptide, e.g., a Streptomyces lividans or Streptomyces murinus
polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli
or a Pseudomonas sp. polypeptide.

[0078] A polypeptide of the present invention may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a Candida,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia
polypeptide; or more preferably a filamentous fungal polypeptide such as
an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,
Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma
polypeptide.

[0081] In another preferred aspect, the polypeptide is a Eurotium
amstelodami, Aspergillus amstelodami, Aspergillus montevidensis or
Aspergillus vitis polypeptide.

[0082] In a more preferred aspect, the polypeptide is a Eurotium
amstelodami polypeptide, e.g., the polypeptide of SEQ ID NO:2.

[0083] It will be understood that for the aforementioned species, the
invention encompasses both the perfect and imperfect states, and other
taxonomic equivalents, e.g., anamorphs, regardless of the species name by
which they are known. Those skilled in the art will readily recognize the
identity of appropriate equivalents.

[0084] Strains of these species are readily accessible to the public in a
number of culture collections, such as the American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen
GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural
Research Service Patent Culture Collection, Northern Regional Research
Center (NRRL).

[0085] Furthermore, such polypeptides may be identified and obtained from
other sources including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) using the above-mentioned probes. Techniques for
isolating microorganisms from natural habitats are well known in the art.
The polynucleotide may then be obtained by similarly screening a genomic
or cDNA library of another microorganism. Once a polynucleotide sequence
encoding a polypeptide has been detected with the probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques which
are well known to those of ordinary skill in the art (see, e.g., Sambrook
et al., 1989, supra).

[0086] Polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding another
polypeptide to a nucleotide sequence (or a portion thereof) of the
present invention. Techniques for producing fusion polypeptides are known
in the art, and include ligating the coding sequences encoding the
polypeptides so that they are in frame and that expression of the fused
polypeptide is under control of the same promoter(s) and terminator.

Polynucleotides

[0087] The present invention also relates to isolated polynucleotides
having a nucleotide sequence which encode a polypeptide of the present
invention. In a preferred aspect, the nucleotide sequence is set forth in
SEQ ID NO:1. In another preferred aspect, the nucleotide sequence is the
mature polypeptide coding region of SEQ ID NO:1. The present invention
also encompasses nucleotide sequences which encode a polypeptide having
the amino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof,
which differ from SEQ ID NO:1 by virtue of the degeneracy of the genetic
code. The present invention also relates to subsequences of SEQ ID NO:1
which encode fragments of SEQ ID NO:2 that have antimicrobial activity.

[0088] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO:1, in which the mutant nucleotide sequence encodes
a polypeptide which consists of amino acids 1 to 42 of SEQ ID NO:2.

[0089] The techniques used to isolate or clone a polynucleotide encoding a
polypeptide are known in the art and include isolation from genomic DNA,
preparation from cDNA, or a combination thereof. The cloning of the
polynucleotides of the present invention from such genomic DNA can be
effected, e.g., by using the well known polymerase chain reaction (PCR)
or antibody screening of expression libraries to detect cloned DNA
fragments with shared structural features. See, e.g., Innis et al., 1990,
PCR: A Guide to Methods and Application, Academic Press, New York. Other
nucleic acid amplification procedures such as ligase chain reaction
(LCR), ligated activated transcription (LAT) and nucleotide
sequence-based amplification (NASBA) may be used. The polynucleotides may
be cloned from a strain of Eurotium, or another or related organism and
thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleotide sequence.

[0090] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO:1 (i.e., nucleotides 145 to 270)
of at least 60%, preferably at least 65%, more preferably at least 70%,
more preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity, which
encode an active polypeptide.

[0091] Modification of a nucleotide sequence encoding a polypeptide of the
present invention may be necessary for the synthesis of polypeptides
substantially similar to the polypeptide. The term "substantially
similar" to the polypeptide refers to non-naturally occurring forms of
the polypeptide. These polypeptides may differ in some engineered way
from the polypeptide isolated from its native source, e.g., artificial
variants that differ in specific activity, thermostability, pH optimum,
or the like. The variant sequence may be constructed on the basis of the
nucleotide sequence presented as the polypeptide encoding region of SEQ
ID NO:1, e.g., a subsequence thereof, and/or by introduction of
nucleotide substitutions which do not give rise to another amino acid
sequence of the polypeptide encoded by the nucleotide sequence, but which
correspond to the codon usage of the host organism intended for
production of the enzyme, or by introduction of nucleotide substitutions
which may give rise to a different amino acid sequence. For a general
description of nucleotide substitution, see, e.g., Ford et al., 1991,
Protein Expression and Purification 2: 95-107.

[0092] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the function of
the molecule and still result in an active polypeptide. Amino acid
residues essential to the activity of the polypeptide encoded by an
isolated polynucleotide of the invention, and therefore preferably not
subject to substitution, may be identified according to procedures known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244:
1081-1085). In the latter technique, mutations are introduced at every
positively charged residue in the molecule, and the resultant mutant
molecules are tested for antimicrobial activity to identify amino acid
residues that are critical to the activity of the molecule. Sites of
substrate-enzyme interaction can also be determined by analysis of the
three-dimensional structure as determined by such techniques as nuclear
magnetic resonance analysis, crystallography or photoaffinity labelling
(see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al.,
1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992,
FEBS Letters 309: 59-64).

[0093] The present invention also relates to isolated polynucleotides
encoding a polypeptide of the present invention, which hybridize under
low stringency conditions, preferably medium stringency conditions, more
preferably medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with (i) nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA
sequence contained in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) a
complementary strand of (i) or (ii); or allelic variants and subsequences
thereof (Sambrook et al., 1989, supra), as defined herein.

[0094] The present invention also relates to isolated polynucleotides
obtained by (a) hybridizing a population of DNA under low, medium,
medium-high, high, or very high stringency conditions with (i)
nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA sequence contained
in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) a complementary strand
of (i) or (ii); and (b) isolating the hybridizing polynucleotide, which
encodes a polypeptide having antimicrobial activity.

Nucleic Acid Constructs

[0095] The present invention also relates to nucleic acid constructs
comprising an isolated polynucleotide of the present invention operably
linked to one or more control sequences which direct the expression of
the coding sequence in a suitable host cell under conditions compatible
with the control sequences.

[0096] An isolated polynucleotide encoding a polypeptide of the present
invention may be manipulated in a variety of ways to provide for
expression of the polypeptide. Manipulation of the polynucleotide's
sequence prior to its insertion into a vector may be desirable or
necessary depending on the expression vector. The techniques for
modifying polynucleotide sequences utilizing recombinant DNA methods are
well known in the art.

[0097] The control sequence may be an appropriate promoter sequence, a
nucleotide sequence which is recognized by a host cell for expression of
a polynucleotide encoding a polypeptide of the present invention. The
promoter sequence contains transcriptional control sequences which
mediate the expression of the polypeptide. The promoter may be any
nucleotide sequence which shows transcriptional activity in the host cell
of choice including mutant, truncated, and hybrid promoters, and may be
obtained from genes encoding extracellular or intracellular polypeptides
either homologous or heterologous to the host cell.

[0101] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to terminate
transcription. The terminator sequence is operably linked to the 3'
terminus of the nucleotide sequence encoding the polypeptide. Any
terminator which is functional in the host cell of choice may be used in
the present invention.

[0104] The control sequence may also be a suitable leader sequence, a
nontranslated region of an mRNA which is important for translation by the
host cell. The leader sequence is operably linked to the 5' terminus of
the nucleotide sequence encoding the polypeptide. Any leader sequence
that is functional in the host cell of choice may be used in the present
invention.

[0107] The control sequence may also be a polyadenylation sequence, a
sequence operably linked to the 3' terminus of the nucleotide sequence
and which, when transcribed, is recognized by the host cell as a signal
to add polyadenosine residues to transcribed mRNA. Any polyadenylation
sequence which is functional in the host cell of choice may be used in
the present invention.

[0110] The control sequence may also be a signal peptide coding region
that codes for an amino acid sequence linked to the amino terminus of a
polypeptide and directs the encoded polypeptide into the cell's secretory
pathway. The 5' end of the coding sequence of the nucleotide sequence may
inherently contain a signal peptide coding region naturally linked in
translation reading frame with the segment of the coding region which
encodes the secreted polypeptide. Alternatively, the 5' end of the coding
sequence may contain a signal peptide coding region which is foreign to
the coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a signal
peptide coding region. Alternatively, the foreign signal peptide coding
region may simply replace the natural signal peptide coding region in
order to enhance secretion of the polypeptide. However, any signal
peptide coding region which directs the expressed polypeptide into the
secretory pathway of a host cell of choice may be used in the present
invention.

[0115] The control sequence may also be a propeptide coding region that
codes for an amino acid sequence positioned at the amino terminus of a
polypeptide. The resultant polypeptide is known as a proenzyme or
propolypeptide (or a zymogen in some cases). A propolypeptide is
generally inactive and can be converted to a mature active polypeptide by
catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide. The propeptide coding region may be obtained from the
genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis
neutral protease (npr7), Saccharomyces cerevisiae alpha-factor,
Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila
laccase (WO 95/33836).

[0116] In a preferred aspect, the propeptide coding region is nucleotides
61 to 144 of SEQ ID NO:1 which encode amino acids -28 to -1 of SEQ ID
NO:2.

[0117] Where both signal peptide and propeptide regions are present at the
amino terminus of a polypeptide, the propeptide region is positioned next
to the amino terminus of a polypeptide and the signal peptide region is
positioned next to the amino terminus of the propeptide region.

[0118] It may also be desirable to add regulatory sequences which allow
the regulation of the expression of the polypeptide relative to the
growth of the host cell. Examples of regulatory systems are those which
cause the expression of the gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a regulatory
compound. Regulatory systems in prokaryotic systems include the lac, tac,
and trp operator systems. In yeast, the ADH2 system or GAL1 system may be
used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter
may be used as regulatory sequences. Other examples of regulatory
sequences are those which allow for gene amplification. In eukaryotic
systems, these include the dihydrofolate reductase gene which is
amplified in the presence of methotrexate, and the metallothionein genes
which are amplified with heavy metals. In these cases, the nucleotide
sequence encoding the polypeptide would be operably linked with the
regulatory sequence.

Expression Vectors

[0119] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a promoter,
and transcriptional and translational stop signals. The various nucleic
acids and control sequences described above may be joined together to
produce a recombinant expression vector which may include one or more
convenient restriction sites to allow for insertion or substitution of
the nucleotide sequence encoding the polypeptide at such sites.
Alternatively, a nucleotide sequence of the present invention may be
expressed by inserting the nucleotide sequence or a nucleic acid
construct comprising the sequence into an appropriate vector for
expression. In creating the expression vector, the coding sequence is
located in the vector so that the coding sequence is operably linked with
the appropriate control sequences for expression.

[0120] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to recombinant DNA
procedures and can bring about expression of the nucleotide sequence. The
choice of the vector will typically depend on the compatibility of the
vector with the host cell into which the vector is to be introduced. The
vectors may be linear or closed circular plasmids.

[0121] The vector may be an autonomously replicating vector, i.e., a
vector which exists as an extrachromosomal entity, the replication of
which is independent of chromosomal replication, e.g., a plasmid, an
extrachromosomal element, a minichromosome, or an artificial chromosome.
The vector may contain any means for assuring self-replication.
Alternatively, the vector may be one which, when introduced into the host
cell, is integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Furthermore, a single
vector or plasmid or two or more vectors or plasmids which together
contain the total DNA to be introduced into the genome of the host cell,
or a transposon may be used.

[0122] The vectors of the present invention preferably contain one or more
selectable markers which permit easy selection of transformed cells. A
selectable marker is a gene the product of which provides for biocide or
viral resistance, resistance to heavy metals, prototrophy to auxotrophs,
and the like.

[0123] Examples of bacterial selectable markers are the daI genes from
Bacillus subtilis or Bacillus licheniformis, or markers which confer
antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or
tetracycline resistance. Suitable markers for yeast host cells are ADE2,
HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a
filamentous fungal host cell include, but are not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase),
and trpC (anthranilate synthase), as well as equivalents thereof.
Preferred for use in an Aspergillus cell are the amdS and pyrG genes of
Aspergillus nidulans or Aspergillus oryzae and the bar gene of
Streptomyces hygroscopicus.

[0124] The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host cell's
genome or autonomous replication of the vector in the cell independent of
the genome.

[0125] For integration into the host cell genome, the vector may rely on
the polynucleotide's sequence encoding the polypeptide or any other
element of the vector for integration into the genome by homologous or
nonhomologous recombination. Alternatively, the vector may contain
additional nucleotide sequences for directing integration by homologous
recombination into the genome of the host cell at a precise location(s)
in the chromosome(s). To increase the likelihood of integration at a
precise location, the integrational elements should preferably contain a
sufficient number of nucleic acids, such as 100 to 10,000 base pairs,
preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000
base pairs, which have a high degree of identity with the corresponding
target sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous with
the target sequence in the genome of the host cell. Furthermore, the
integrational elements may be non-encoding or encoding nucleotide
sequences. On the other hand, the vector may be integrated into the
genome of the host cell by non-homologous recombination.

[0126] For autonomous replication, the vector may further comprise an
origin of replication enabling the vector to replicate autonomously in
the host cell in question. The origin of replication may be any plasmid
replicator mediating autonomous replication which functions in a cell.
The term "origin of replication" or "plasmid replicator" is defined
herein as a nucleotide sequence that enables a plasmid or vector to
replicate in vivo.

[0127] Examples of bacterial origins of replication are the origins of
replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting
replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1
permitting replication in Bacillus.

[0128] Examples of origins of replication for use in a yeast host cell are
the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1
and CEN3, and the combination of ARS4 and CEN6.

[0129] Examples of origins of replication useful in a filamentous fungal
cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,
1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of
the AMA1 gene and construction of plasmids or vectors comprising the gene
can be accomplished according to the methods disclosed in WO 00/24883.

[0130] More than one copy of a polynucleotide of the present invention may
be inserted into the host cell to increase production of the gene
product. An increase in the copy number of the polynucleotide can be
obtained by integrating at least one additional copy of the sequence into
the host cell genome or by including an amplifiable selectable marker
gene with the polynucleotide where cells containing amplified copies of
the selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.

[0131] The procedures used to ligate the elements described above to
construct the recombinant expression vectors of the present invention are
well known to one skilled in the art (see, e.g., Sambrook et al., 1989,
supra).

Host Cells

[0132] The present invention also relates to recombinant host cells,
comprising a polynucleotide of the present invention, which are
advantageously used in the recombinant production of the polypeptides. A
vector comprising a polynucleotide of the present invention is introduced
into a host cell so that the vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as described
earlier. The term "host cell" encompasses any progeny of a parent cell
that is not identical to the parent cell due to mutations that occur
during replication. The choice of a host cell will to a large extent
depend upon the gene encoding the polypeptide and its source.

[0133] The host cell may be a unicellular microorganism, e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.

[0136] The host cell may also be a eukaryote, such as a mammalian, insect,
plant, or fungal cell.

[0137] In a preferred aspect, the host cell is a fungal cell. "Fungi" as
used herein includes the phyla Ascomycota, Basidiomycota,
Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,
Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the Oomycota
(as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic
fungi (Hawksworth et al., 1995, supra).

[0138] In a more preferred aspect, the fungal host cell is a yeast cell.
"Yeast" as used herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti
(Blastomycetes). Since the classification of yeast may change in the
future, for the purposes of this invention, yeast shall be defined as
described in Biology and Activities of Yeast (Skinner, F. A., Passmore,
S. M., and Davenport, R. R., eds, Soc. App. Bacterial, Symposium Series
No. 9, 1980).

[0139] In an even more preferred aspect, the yeast host cell is a Candida,
Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell.

[0140] In a most preferred aspect, the yeast host cell is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,
Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis
or Saccharomyces oviformis cell. In another most preferred aspect, the
yeast host cell is a Kluyveromyces lactis cell. In another most preferred
aspect, the yeast host cell is a Yarrowia lipolytica cell.

[0141] In another more preferred aspect, the fungal host cell is a
filamentous fungal cell. "Filamentous fungi" include all filamentous
forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth
et al., 1995, supra). The filamentous fungi are generally characterized
by a mycelial wall composed of chitin, cellulose, glucan, chitosan,
mannan, and other complex polysaccharides. Vegetative growth is by hyphal
elongation and carbon catabolism is obligately aerobic. In contrast,
vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be
fermentative.

[0144] Fungal cells may be transformed by a process involving protoplast
formation, transformation of the protoplasts, and regeneration of the
cell wall in a manner known per se. Suitable procedures for
transformation of Aspergillus and Trichoderma host cells are described in
EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy
of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium
species are described by Malardier et al., 1989, Gene 78: 147-156, and WO
96/00787. Yeast may be transformed using the procedures described by
Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide
to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume
194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of
the National Academy of Sciences USA 75: 1920.

Methods of Production

[0145] The present invention also relates to methods for producing a
polypeptide of the present invention, comprising (a) cultivating a cell,
which in its wild-type form is capable of producing the polypeptide,
under conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide. Preferably, the cell is of the genus
Eurotium, and more preferably Eurotium amstelodami.

[0146] The present invention also relates to methods for producing a
polypeptide of the present invention, comprising (a) cultivating a host
cell under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.

[0147] The present invention also relates to methods for producing a
polypeptide of the present invention, comprising (a) cultivating a host
cell under conditions conducive for production of the polypeptide,
wherein the host cell comprises a mutant nucleotide sequence having at
least one mutation in the mature polypeptide coding region of SEQ ID
NO:1, wherein the mutant nucleotide sequence encodes a polypeptide which
consists of amino acids 1 to 42 of SEQ ID NO:2, and (b) recovering the
polypeptide.

[0148] In the production methods of the present invention, the cells are
cultivated in a nutrient medium suitable for production of the
polypeptide using methods well known in the art. For example, the cell
may be cultivated by shake flask cultivation, and small-scale or
large-scale fermentation (including continuous, batch, fed-batch, or
solid state fermentations) in laboratory or industrial fermentors
performed in a suitable medium and under conditions allowing the
polypeptide to be expressed and/or isolated. The cultivation takes place
in a suitable nutrient medium comprising carbon and nitrogen sources and
inorganic salts, using procedures known in the art. Suitable media are
available from commercial suppliers or may be prepared according to
published compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient medium, the
polypeptide can be recovered directly from the medium. If the polypeptide
is not secreted, it can be recovered from cell lysates.

[0149] The polypeptides may be detected using methods known in the art
that are specific for the polypeptides. These detection methods may
include use of specific antibodies. For example, an antimicrobial
activity assay may be used to determine the activity of the polypeptide
as described herein.

[0150] The resulting polypeptide may be recovered using methods known in
the art. For example, the polypeptide may be recovered from the nutrient
medium by conventional procedures including, but not limited to,
centrifugation, filtration, extraction, spray-drying, evaporation, or
precipitation.

[0152] The present invention also relates to a transgenic plant, plant
part, or plant cell which has been transformed with a nucleotide sequence
encoding a polypeptide having antimicrobial activity of the present
invention so as to express and produce the polypeptide in recoverable
quantities. The polypeptide may be recovered from the plant or plant
part. Alternatively, the plant or plant part containing the recombinant
polypeptide may be used as such for improving the quality of a food or
feed, e.g., improving nutritional value, palatability, and rheological
properties, or to destroy an antinutritive factor.

[0155] Examples of plant parts are stem, callus, leaves, root, fruits,
seeds, and tubers as well as the individual tissues comprising these
parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues,
meristems. Specific plant cell compartments, such as chloroplasts,
apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also
considered to be a plant part. Furthermore, any plant cell, whatever the
tissue origin, is considered to be a plant part. Likewise, plant parts
such as specific tissues and cells isolated to facilitate the utilisation
of the invention are also considered plant parts, e.g., embryos,
endosperms, aleurone and seeds coats.

[0156] Also included within the scope of the present invention are the
progeny of such plants, plant parts, and plant cells.

[0157] The transgenic plant or plant cell expressing a polypeptide of the
present invention may be constructed in accordance with methods known in
the art. In short, the plant or plant cell is constructed by
incorporating one or more expression constructs encoding a polypeptide of
the present invention into the plant host genome and propagating the
resulting modified plant or plant cell into a transgenic plant or plant
cell.

[0158] The expression construct is conveniently a nucleic acid construct
which comprises a polynucleotide encoding a polypeptide of the present
invention operably linked with appropriate regulatory sequences required
for expression of the nucleotide sequence in the plant or plant part of
choice. Furthermore, the expression construct may comprise a selectable
marker useful for identifying host cells into which the expression
construct has been integrated and DNA sequences necessary for
introduction of the construct into the plant in question (the latter
depends on the DNA introduction method to be used).

[0159] The choice of regulatory sequences, such as promoter and terminator
sequences and optionally signal or transit sequences is determined, for
example, on the basis of when, where, and how the polypeptide is desired
to be expressed. For instance, the expression of the gene encoding a
polypeptide of the present invention may be constitutive or inducible, or
may be developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or leaves.
Regulatory sequences are, for example, described by Tague et al., 1988,
Plant Physiology 86: 506.

[0161] A promoter enhancer element may also be used to achieve higher
expression of a polypeptide of the present invention in the plant. For
instance, the promoter enhancer element may be an intron which is placed
between the promoter and the nucleotide sequence encoding a polypeptide
of the present invention. For instance, Xu et al., 1993, supra, disclose
the use of the first intron of the rice actin 1 gene to enhance
expression.

[0162] The selectable marker gene and any other parts of the expression
construct may be chosen from those available in the art.

[0164] Presently, Agrobacterium tumefaciens-mediated gene transfer is the
method of choice for generating transgenic dicots (for a review, see
Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and
can also be used for transforming monocots, although other transformation
methods are often used for these plants. Presently, the method of choice
for generating transgenic monocots is particle bombardment (microscopic
gold or tungsten particles coated with the transforming DNA) of embryonic
calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281;
Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al.,
1992, Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.

[0165] Following transformation, the transformants having incorporated the
expression construct are selected and regenerated into whole plants
according to methods well-known in the art. Often the transformation
procedure is designed for the selective elimination of selection genes
either during regeneration or in the following generations by using, for
example, co-transformation with two separate T-DNA constructs or site
specific excision of the selection gene by a specific recombinase.

[0166] The present invention also relates to methods for producing a
polypeptide of the present invention comprising (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide encoding a
polypeptide having antimicrobial activity of the present invention under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.

Compositions

[0167] The present invention also relates to compositions, such as
pharmaceutical compositions, comprising a polypeptide of the present
invention. Preferably, the compositions are enriched in such a
polypeptide. The term "enriched" indicates that the antimicrobial
activity of the composition has been increased, e.g., with an enrichment
factor of 1.1.

[0169] In an embodiment the biocidal agent is a non-enzymatic chemical
agent. In another embodiment the biocidal agent is a non-polypeptide
chemical agent.

[0170] The compositions may comprise a suitable carrier material. The
compositions may also comprise a suitable delivery vehicle capable of
delivering the antimicrobial polypeptides of the invention to the desired
locus when the compositions are used as a medicament.

[0171] The polypeptide compositions may be prepared in accordance with
methods known in the art and may be in the form of a liquid or a dry
composition. For instance, the polypeptide composition may be in the form
of a granulate or a microgranulate. The polypeptide to be included in the
composition may be stabilized in accordance with methods known in the
art.

[0172] Examples are given below of preferred uses of the polypeptide
compositions of the invention. The dosage of the polypeptide composition
of the invention and other conditions under which the composition is used
may be determined on the basis of methods known in the art.

Methods and Uses

[0173] The present invention is also directed to methods for using the
polypeptides having antimicrobial activity. The antimicrobial
polypeptides are typically useful at any locus subject to contamination
by bacteria, fungi, yeast or algae. Typically, loci are in aqueous
systems such as cooling water systems, laundry rinse water, oil systems
such as cutting oils, lubricants, oil fields and the like, where
microorganisms need to be killed or where their growth needs to be
controlled. However, the present invention may also be used in all
applications for which known antimicrobial compositions are useful, such
as protection of wood, latex, adhesive, glue, paper, cardboard, textile,
leather, plastics, caulking, and feed.

[0175] Thus, the antimicrobial polypeptides of the invention may by useful
as a disinfectant, e.g., in the treatment of infections in the eye or the
mouth, skin infections; in antiperspirants or deodorants; for cleaning
and disinfection of contact lenses and teeth (oral care).

[0176] In general it is contemplated that the antimicrobial polypeptides
of the present invention are useful for cleaning, disinfecting or
inhibiting microbial growth on any surface. Examples of surfaces, which
may advantageously be contacted with the antimicrobial polypeptides of
the invention are surfaces of process equipment used e.g. dairies,
chemical or pharmaceutical process plants, water sanitation systems, oil
processing plants, paper pulp processing plants, water treatment plants,
and cooling towers. The antimicrobial polypeptides of the invention
should be used in an amount, which is effective for cleaning,
disinfecting or inhibiting microbial growth on the surface in question.

[0177] The antimicrobial polypeptides of the invention may additionally be
used for cleaning surfaces and cooking utensils in food processing plants
and in any area in which food is prepared or served such as hospitals,
nursing homes, and restaurants.

[0178] It may also be used as a preservation agent or a disinfection agent
in water based paints.

[0179] The invention also relates to the use of an antimicrobial
polypeptide or composition of the invention as a medicament. Further, an
antimicrobial polypeptide or composition of the invention may also be
used for the manufacture of a medicament for controlling or combating
microorganisms, such as fungal organisms or bacteria, preferably gram
positive bacteria.

[0180] The composition and antimicrobial polypeptide of the invention may
be used as an antimicrobial veterinarian or human therapeutic or
prophylactic agent. Thus, the composition and antimicrobial polypeptide
of the invention may be used in the preparation of veterinarian or human
therapeutic agents or prophylactic agents for the treatment of microbial
infections, such as bacterial or fungal infections, preferably gram
positive bacterial infections. In particular the microbial infections may
be associated with lung diseases including, but not limited to,
tuberculosis, pneumonia and cystic fibrosis; and sexual transmitted
diseases including, but not limited to, gonorrhea and chlamydia.

[0181] The composition of the invention comprises an effective amount of
the antimicrobial polypeptide of the invention.

[0182] The term "effective amount" when used herein is intended to mean an
amount of the antimicrobial polypeptides of the invention, which is
sufficient to inhibit growth of the microorganisms in question.

[0183] The invention also relates to wound healing compositions or
products such as bandages, medical devices such as, e.g., catheters and
further to anti-dandruff hair products, such as shampoos.

[0184] Formulations of the antimicrobial polypeptides of the invention are
administered to a host suffering from or predisposed to a microbial
infection. Administration may be topical, localized or systemic,
depending on the specific microorganism, preferably it will be localized.
Generally the dose of the antimicrobial polypeptides of the invention
will be sufficient to decrease the microbial population by at least about
50%, usually by at least 1 log, and may be by 2 or more logs of killing.
The compounds of the present invention are administered at a dosage that
reduces the microbial population while minimizing any side-effects. It is
contemplated that the composition will be obtained and used under the
guidance of a physician for in vivo use. The antimicrobial polypeptides
of the invention are particularly useful for killing gram negative
bacteria, including Pseudomonas aeruginosa, and Chlamydia trachomatis;
and gram-positive bacteria, including streptococci such as Streptococcus
pneumonia, S. uberis, S. hyointestinalis, S. pyogenes or agalactiae; and
staphylococci such as Staphylococcus aureus, S. epidermidis, S. simulans,
S. xylosus, S. camosus.

[0185] Formulations of the antimicrobial polypeptides of the invention may
be administered to a host suffering from or predisposed to a microbial
lung infection, such as pneumonia; or to a microbial wound infection,
such as a bacterial wound infection.

[0186] Formulations of the antimicrobial polypeptides of the invention may
also be administered to a host suffering from or predisposed to a skin
infection, such as acne, atopic dermatitis or seborrheic dermatitis;
preferably the skin infection is a bacterial skin infection, e.g. caused
by Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium
acnes, Pityrosporum ovale or Malassezia furfur.

[0187] The antimicrobial polypeptides of the invention are also useful for
in vitro formulations to kill microbes, particularly where one does not
wish to introduce quantities of conventional antibiotics. For example,
the antimicrobial polypeptides of the invention may be added to animal
and/or human food preparations; or they may be included as an additive
for in vitro cultures of cells, to prevent the overgrowth of microbes in
tissue culture.

[0188] The susceptibility of a particular microbe to killing with the
antimicrobial polypeptides of the invention may be determined by in vitro
testing, as detailed in the experimental section. Typically a culture of
the microbe is combined with the antimicrobial polypeptide at varying
concentrations for a period of time sufficient to allow the protein to
act, usually between about one hour and one day. The viable microbes are
then counted, and the level of killing determined.

[0191] Various methods for administration may be employed. The polypeptide
formulation may be given orally, or may be injected intravascularly,
subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder,
topically, etc. For example, methods of administration by inhalation are
well-known in the art. The dosage of the therapeutic formulation will
vary widely, depending on the specific antimicrobial polypeptide to be
administered, the nature of the disease, the frequency of administration,
the manner of administration, the clearance of the agent from the host,
and the like. The initial dose may be larger, followed by smaller
maintenance doses. The dose may be administered as infrequently as weekly
or biweekly, or fractionated into smaller doses and administered once or
several times daily, semi-weekly, etc. to maintain an effective dosage
level. In many cases, oral administration will require a higher dose than
if administered intravenously. The amide bonds, as well as the amino and
carboxy termini, may be modified for greater stability on oral
administration. For example, the carboxy terminus may be amidated.

Formulations

[0192] The compounds of this invention can be incorporated into a variety
of formulations for therapeutic administration. More particularly, the
compounds of the present invention can be formulated into pharmaceutical
compositions by combination with appropriate, pharmaceutically acceptable
carriers or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,
granules, ointments, creams, foams, solutions, suppositories, injections,
inhalants, gels, microspheres, lotions, and aerosols. As such,
administration of the compounds can be achieved in various ways,
including oral, buccal, rectal, parenteral, intraperitoneal, intradermal,
transdermal, intracheal, etc., administration. The antimicrobial
polypeptides of the invention may be systemic after administration or may
be localized by the use of an implant or other formulation that acts to
retain the active dose at the site of implantation.

[0193] In one embodiment, a formulation for topical use comprises a
chelating agent that decreases the effective concentration of divalent
cations, particularly calcium and magnesium. For example, agents such as
citrate, EGTA or EDTA may be included, where citrate is preferred. The
concentration of citrate will usually be from about 1 to 10 mM.

[0194] The compounds of the present invention can be administered alone,
in combination with each other, or they can be used in combination with
other known compounds (e.g., perforin, anti-inflammatory agents,
antibiotics, etc.) In pharmaceutical dosage forms, the compounds may be
administered in the form of their pharmaceutically acceptable salts. The
following methods and excipients are merely exemplary and are in no way
limiting.

[0195] For oral preparations, the compounds can be used alone or in
combination with appropriate additives to make tablets, powders, granules
or capsules, for example, with conventional additives, such as lactose,
mannitol, corn starch or potato starch; with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch or gelatins; with
disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants, such as talc or magnesium
stearate; and if desired, with diluents, buffering agents, moistening
agents, preservatives and flavoring agents.

[0196] The compounds can be formulated into preparations for injections by
dissolving, suspending or emulsifying them in an aqueous or nonaqueous
solvent, such as vegetable or other similar oils, synthetic aliphatic
acid glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers and
preservatives.

[0197] The compounds can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention can
be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.

[0198] The compounds can be used as lotions, for example to prevent
infection of burns, by formulation with conventional additives such as
solubilizers, isotonic agents, suspending agents, emulsifying agents,
stabilizers and preservatives.

[0199] Furthermore, the compounds can be made into suppositories by mixing
with a variety of bases such as emulsifying bases or water-soluble bases.
The compounds of the present invention can be administered rectally via a
suppository. The suppository can include vehicles such as cocoa butter,
carbowaxes and polyethylene glycols, which melt at body temperature, yet
are solidified at room temperature.

[0200] Unit dosage forms for oral or rectal administration such as syrups,
elixirs, and suspensions may be provided wherein each dosage unit, for
example, teaspoonful, tablespoonful, tablet or suppository, contains a
predetermined amount of the composition containing one or more compounds
of the present invention. Similarly, unit dosage forms for injection or
intravenous administration may comprise the compound of the present
invention in a composition as a solution in sterile water, normal saline
or another pharmaceutically acceptable carrier.

[0201] Implants for sustained release formulations are well-known in the
art. Implants are formulated as microspheres, slabs, etc. with
biodegradable or non-biodegradable polymers. For example, polymers of
lactic acid and/or glycolic acid form an erodible polymer that is
well-tolerated by the host. The implant containing the antimicrobial
polypeptides of the invention is placed in proximity to the site of
infection, so that the local concentration of active agent is increased
relative to the rest of the body.

[0202] The term "unit dosage form", as used herein, refers to physically
discrete units suitable as unitary dosages for human and animal subjects,
each unit containing a predetermined quantity of compounds of the present
invention calculated in an amount sufficient to produce the desired
effect in association with a pharmaceutically acceptable diluent, carrier
or vehicle. The specifications for the unit dosage forms of the present
invention depend on the particular compound employed and the effect to be
achieved, and the pharmacodynamics associated with the compound in the
host.

[0203] The pharmaceutically acceptable excipients, such as vehicles,
adjuvants, carriers or diluents, are readily available to the public.
Moreover, pharmaceutically acceptable auxiliary substances, such as pH
adjusting and buffering agents, tonicity adjusting agents, stabilizers,
wetting agents and the like, are readily available to the public.

[0204] Typical dosages for systemic administration range from 0.1 pg to
100 milligrams per kg weight of subject per administration. A typical
dosage may be one tablet taken from two to six times daily, or one
time-release capsule or tablet taken once a day and containing a
proportionally higher content of active ingredient. The time-release
effect may be obtained by capsule materials that dissolve at different pH
values, by capsules that release slowly by osmotic pressure, or by any
other known means of controlled release.

[0205] Those of skill will readily appreciate that dose levels can vary as
a function of the specific compound, the severity of the symptoms and the
susceptibility of the subject to side effects. Some of the specific
compounds are more potent than others. Preferred dosages for a given
compound are readily determinable by those of skill in the art by a
variety of means. A preferred means is to measure the physiological
potency of a given compound.

[0206] The use of liposomes as a delivery vehicle is one method of
interest. The liposomes fuse with the cells of the target site and
deliver the contents of the lumen intracellularly. The liposomes are
maintained in contact with the cells for sufficient time for fusion,
using various means to maintain contact, such as isolation, binding
agents, and the like. In one aspect of the invention, liposomes are
designed to be aerosolized for pulmonary administration. Liposomes may be
prepared with purified proteins or peptides that mediate fusion of
membranes, such as Sendai virus or influenza virus, etc. The lipids may
be any useful combination of known liposome forming lipids, including
cationic or zwitterionic lipids, such as phosphatidylcholine. The
remaining lipid will be normally be neutral or acidic lipids, such as
cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

[0207] For preparing the liposomes, the procedure described by Kato et
al., (1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids and
lumen composition containing peptides are combined in an appropriate
aqueous medium, conveniently a saline medium where the total solids will
be in the range of about 1-10 weight percent. After intense agitation for
short periods of time, from about 5-60 sec., the tube is placed in a warm
water bath, from about 25-40° C. and this cycle repeated from
about 5-10 times. The composition is then sonicated for a convenient
period of time, generally from about 1-10 sec. and may be further
agitated by vortexing. The volume is then expanded by adding aqueous
medium, generally increasing the volume by about from 1-2 fold, followed
by shaking and cooling. This method allows for the incorporation into the
lumen of high molecular weight molecules.

[0209] Anti-mycotic agents are also useful, including polyenes, e.g.
amphotericin B, nystatin; 5-flucosyn; and azoles, e.g. miconazol,
ketoconazol, itraconazol and fluconazol. Antituberculotic drugs include
isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be
included in a formulation of the antimicrobial polypeptides of the
invention, e.g. interferon gamma, tumor necrosis factor alpha,
interleukin 12, etc.

In Vitro Synthesis

[0210] The antimicrobial peptides of the invention may be prepared by in
vitro synthesis, using conventional methods as known in the art. Various
commercial synthetic apparatuses are available, for example automated
synthesizers by Applied Biosystems Inc., Beckman, etc. By using
synthesizers, naturally occurring amino acids may be substituted with
unnatural amino acids, particularly D-isomers (or D-forms) e.g. D-alanine
and D-isoleucine, diastereoisomers, side chains having different lengths
or functionalities, and the like. The particular sequence and the manner
of preparation will be determined by convenience, economics, purity
required, and the like.

[0211] Chemical linking may be provided to various peptides or proteins
comprising convenient functionalities for bonding, such as amino groups
for amide or substituted amine formation, e.g. reductive amination, thiol
groups for thioether or disulfide formation, carboxyl groups for amide
formation, and the like.

[0212] If desired, various groups may be introduced into the peptide
during synthesis or during expression, which allow for linking to other
molecules or to a surface. Thus cysteines can be used to make thioethers,
histidines for linking to a metal ion complex, carboxyl groups for
forming amides or esters, amino groups for forming amides, and the like.

[0213] The polypeptides may also be isolated and purified in accordance
with conventional methods of recombinant synthesis. A lysate may be
prepared of the expression host and the lysate purified using HPLC,
exclusion chromatography, gel electrophoresis, affinity chromatography,
or other purification technique. For the most part, the compositions
which are used will comprise at least 20% by weight of the desired
product, more usually at least about 75% by weight, preferably at least
about 95% by weight, and for therapeutic purposes, usually at least about
99.5% by weight, in relation to contaminants related to the method of
preparation of the product and its purification. Usually, the percentages
will be based upon total protein

Animal Feed

[0214] The present invention is also directed to methods for using the
polypeptides having antimicrobial activity in animal feed, as well as to
feed compositions and feed additives comprising the antimicrobial
polypeptides of the invention.

[0215] The term animal includes all animals, including human beings.
Examples of animals are non-ruminants, and ruminants, such as cows, sheep
and horses. In a particular embodiment, the animal is a non-ruminant
animal. Non-ruminant animals include mono-gastric animals, e.g. pigs or
swine (including, but not limited to, piglets, growing pigs, and sows);
poultry such as turkeys and chicken (including but not limited to broiler
chicks, layers); young calves; and fish (including but not limited to
salmon).

[0216] The term feed or feed composition means any compound, preparation,
mixture, or composition suitable for, or intended for intake by an
animal.

[0217] In the use according to the invention the antimicrobial polypeptide
can be fed to the animal before, after, or simultaneously with the diet.
The latter is preferred.

[0218] In a particular embodiment, the antimicrobial polypeptide, in the
form in which it is added to the feed, or when being included in a feed
additive, is well defined. Well-defined means that the antimicrobial
polypeptide preparation is at least 50% pure as determined by
Size-exclusion chromatography (see Example 12 of WO 01/58275). In other
particular embodiments the antimicrobial polypeptide preparation is at
least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined
by this method.

[0219] A well-defined antimicrobial polypeptide preparation is
advantageous. For instance, it is much easier to dose correctly to the
feed an antimicrobial polypeptide that is essentially free from
interfering or contaminating other antimicrobial polypeptides. The term
dose correctly refers in particular to the objective of obtaining
consistent and constant results, and the capability of optimising dosage
based upon the desired effect.

[0220] For the use in animal feed, however, the antimicrobial polypeptide
need not be that pure; it may e.g. include other enzymes, in which case
it could be termed an antimicrobial polypeptide preparation.

[0221] The antimicrobial polypeptide preparation can be (a) added directly
to the feed (or used directly in a treatment process of vegetable
proteins), or (b) it can be used in the production of one or more
intermediate compositions such as feed additives or premixes that is
subsequently added to the feed (or used in a treatment process). The
degree of purity described above refers to the purity of the original
antimicrobial polypeptide preparation, whether used according to (a) or
(b) above.

[0222] Antimicrobial polypeptide preparations with purities of this order
of magnitude are in particular obtainable using recombinant methods of
production, whereas they are not so easily obtained and also subject to a
much higher batch-to-batch variation when the antimicrobial polypeptide
is produced by traditional fermentation methods.

[0223] Such antimicrobial polypeptide preparation may of course be mixed
with other enzymes.

[0224] The term vegetable proteins as used herein refers to any compound,
composition, preparation or mixture that includes at least one protein
derived from or originating from a vegetable, including modified proteins
and protein-derivatives. In particular embodiments, the protein content
of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60% (w/w).

[0225] Vegetable proteins may be derived from vegetable protein sources,
such as legumes and cereals, for example materials from plants of the
families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and
Poaceae, such as soy bean meal, lupin meal and rapeseed meal.

[0226] In a particular embodiment, the vegetable protein source is
material from one or more plants of the family Fabaceae, e.g. soybean,
lupine, pea, or bean.

[0227] In another particular embodiment, the vegetable protein source is
material from one or more plants of the family Chenopodiaceae, e.g. beet,
sugar beet, spinach or quinoa.

[0228] Other examples of vegetable protein sources are rapeseed, and
cabbage.

[0229] Soybean is a preferred vegetable protein source.

[0230] Other examples of vegetable protein sources are cereals such as
barley, wheat, rye, oat, maize (corn), rice, and sorghum.

[0231] The antimicrobial polypeptide can be added to the feed in any form,
be it as a relatively pure antimicrobial polypeptide, or in admixture
with other components intended for addition to animal feed, i.e. in the
form of animal feed additives, such as the so-called pre-mixes for animal
feed.

[0232] In a further aspect the present invention relates to compositions
for use in animal feed, such as animal feed, and animal feed additives,
e.g. premixes.

[0233] Apart from the antimicrobial polypeptide of the invention, the
animal feed additives of the invention contain at least one fat soluble
vitamin, and/or at least one water soluble vitamin, and/or at least one
trace mineral, and/or at least one macro mineral.

[0237] Examples of other antifungal polypeptides (AFP's) are the
Aspergillus giganteus, and Aspergillus niger peptides, as well as
variants and fragments thereof which retain antifungal activity, as
disclosed in WO 94/01459 and WO 02/090384.

[0238] Usually fat and water soluble vitamins, as well as trace minerals
form part of a so-called premix intended for addition to the feed,
whereas macro minerals are usually separately added to the feed. Either
of these composition types, when enriched with an antimicrobial
polypeptide of the invention, is an animal feed additive of the
invention.

[0239] In a particular embodiment, the animal feed additive of the
invention is intended for being included (or prescribed as having to be
included) in animal diets or feed at levels of 0.01 to 10.0%; more
particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g
feed). This is so in particular for premixes.

[0240] The following are non-exclusive lists of examples of these
components:

[0245] The nutritional requirements of these components (exemplified with
poultry and piglets/pigs) are listed in Table A of WO 01/58275.
Nutritional requirement means that these components should be provided in
the diet in the concentrations indicated.

[0246] In the alternative, the animal feed additive of the invention
comprises at least one of the individual components specified in Table A
of WO 01/58275. At least one means either of, one or more of, one, or
two, or three, or four and so forth up to all thirteen, or up to all
fifteen individual components. More specifically, this at least one
individual component is included in the additive of the invention in such
an amount as to provide an in-feed-concentration within the range
indicated in column four, or column five, or column six of Table A.

[0247] The present invention also relates to animal feed compositions.
Animal feed compositions or diets have a relatively high content of
protein. Poultry and pig diets can be characterised as indicated in Table
B of WO 01/58275, columns 2-3. Fish diets can be characterised as
indicated in column 4 of this Table B. Furthermore such fish diets
usually have a crude fat content of 200-310 g/kg.

[0248] An animal feed composition according to the invention has a crude
protein content of 50-800 g/kg, and furthermore comprises at least one
antimicrobial polypeptide as claimed herein.

[0249] Furthermore, or in the alternative (to the crude protein content
indicated above), the animal feed composition of the invention has a
content of metabolisable energy of 10-30 MJ/kg; and/or a content of
calcium of 0.1-200 g/kg; and/or a content of available phosphorus of
0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a
content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of
lysine of 0.5-50 g/kg.

[0254] In a particular embodiment, the animal feed composition of the
invention contains at least one vegetable protein or protein source as
defined above.

[0255] In still further particular embodiments, the animal feed
composition of the invention contains 0-80% maize; and/or 0-80% sorghum;
and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40%
soybean meal; and/or 0-10% fish meal; and/or 0-20% whey. Animal diets can
e.g. be manufactured as mash feed (non pelleted) or pelleted feed.
Typically, the milled feed-stuffs are mixed and sufficient amounts of
essential vitamins and minerals are added according to the specifications
for the species in question. Enzymes can be added as solid or liquid
enzyme formulations. For example, a solid enzyme formulation is typically
added before or during the mixing step; and a liquid enzyme preparation
is typically added after the pelleting step. The enzyme may also be
incorporated in a feed additive or premix.

[0256] The final enzyme concentration in the diet is within the range of
0.01-200 mg enzyme protein per kg diet, for example in the range of 5-30
mg enzyme protein per kg animal diet.

[0257] The antimicrobial polypeptide may be administered in one or more of
the following amounts (dosage ranges): 0.01-200; or 0.01-100; or
0.05-100; or 0.05-50; or 0.10-10--all these ranges being in mg
antimicrobial polypeptide protein per kg feed (ppm).

[0258] For determining mg antimicrobial polypeptide protein per kg feed,
the antimicrobial polypeptide is purified from the feed composition, and
the specific activity of the purified antimicrobial polypeptide is
determined using a relevant assay (see under antimicrobial activity,
substrates, and assays). The antimicrobial activity of the feed
composition as such is also determined using the same assay, and on the
basis of these two determinations, the dosage in mg antimicrobial
polypeptide protein per kg feed is calculated.

[0259] The same principles apply for determining mg antimicrobial
polypeptide protein in feed additives. Of course, if a sample is
available of the antimicrobial polypeptide used for preparing the feed
additive or the feed, the specific activity is determined from this
sample (no need to purify the antimicrobial polypeptide from the feed
composition or the additive).

Signal Peptide and Propeptide

[0260] The present invention also relates to nucleic acid constructs
comprising a gene encoding a protein operably linked to one or both of a
first nucleotide sequence consisting of nucleotides 1 to 60 of SEQ ID
NO:1 encoding a signal peptide consisting of amino acids -48 to -29 of
SEQ ID NO:2 and a second nucleotide sequence consisting of nucleotides 61
to 144 of SEQ ID NO:1 encoding a propeptide consisting of amino acids -28
to -1 of SEQ ID NO:2, wherein the gene is foreign to the first and second
nucleotide sequences.

[0262] The present invention also relates to methods for producing a
protein comprising (a) cultivating such a recombinant host cell under
conditions suitable for production of the protein; and (b) recovering the
protein.

[0263] The first and second nucleotide sequences may be operably linked to
foreign genes individually with other control sequences or in combination
with other control sequences. Such other control sequences are described
supra. As described earlier, where both signal peptide and propeptide
regions are present at the amino terminus of a protein, the propeptide
region is positioned next to the amino terminus of a protein and the
signal peptide region is positioned next to the amino terminus of the
propeptide region.

[0264] The protein may be native or heterologous to a host cell. The term
"protein" is not meant herein to refer to a specific length of the
encoded product and, therefore, encompasses peptides, oligopeptides, and
proteins. The term "protein" also encompasses two or more polypeptides
combined to form the encoded product. The proteins also include hybrid
polypeptides which comprise a combination of partial or complete
polypeptide sequences obtained from at least two different proteins
wherein one or more may be heterologous or native to the host cell.
Proteins further include naturally occurring allelic and engineered
variations of the above mentioned proteins and hybrid proteins.

[0266] The gene may be obtained from any prokaryotic, eukaryotic, or other
source.

[0267] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.

EXAMPLES

[0268] Chemicals used as buffers and substrates were commercial products
of at least reagent grade. In the following examples, the antimicrobial
polypeptide shown as amino acids 1 to 42 of SEQ ID NO:2 is referred to as
"Eurocin".

Example 1

cDNA library of Eurotium amstelodami

[0269] A cDNA library was prepared from E. amstelodami grown for 5 days on
Potato dextrose agar (PDA). PolyA enriched RNA was purified, cDNA was
synthesized and the library made according to standard molecular biology
procedures. A detailed protocol on the general process can be found in
the examples of international patent application WO 01/12794. The vector
used for cloning was pMhas5, which is shown in SEQ ID NO:8, and which has
the following features:

[0270] The Eurocin encoding nucleotide sequence was amplified from the
cDNA library (see Example 1) in the following manner: 1 microliter of
cDNA (approximately 10 nanogram of DNA) was used as template in a PCR
reaction with Primer A and Primer B.

[0271] 10 pmole of each primer was used in a 50 microliter reaction
volume. Annealing temperature was 55 degrees Celsius and extension at 72
degrees Celsius for 1 minute. A total of 35 cycles were run. The Expand
High Fidelity PCR System (Roche) was used.

[0272] Aliquots of the PCR reaction were separated on a 4% agarose gel. A
distinct band was seen at a size of approximately 300 bp. The PCR
fragment was digested with BamH1 and Xho1 which cut in the overhangs
introduced by the PCR primers. The digested fragments were isolated and
cloned into BamH1-Xho1 digested pMT2786, an Aspergillus expression
plasmid based on the plasmid pMT2188, which is based on pCaHj527 (se
Example 7 of international patent application WO 02/12472; and WO
00/70064). The sequence of the insert was verified to encode the Eurocin
sequence as identified above. The sequence of the PCR fragment is shown
as SEQ ID NO:6.

[0273] The Aspergillus expression plasmid with the PCR product insert was
named pMT2935 and is shown as SEQ ID NO:7.

Example 3

Expression of Eurocin in Aspergillus

[0274] pMT2935 (see SEQ ID NO:7) was transformed into Aspergillus oryzae
strain BECh2 (see international patent application WO 00/39322). 30
transformants were re-isolated twice under selective and non-inducing
conditions on Cove minimal plates with sucrose and acetamide. To test
expression of Eurocin, transformants were grown for 6 days at 30 degrees
celcius in tubes with 10 ml YPM (2% peptone, 1% yeast extract, 2%
maltose). Supernatants were run on NuPage 10% Bis-Tris SDS gels
(Invitrogen) as recommended by the manufacturer with MES running buffer
to allow separation in the low Mw range. The Aspergillus oryzae
transformants grew well even when induced for the expression of Eurocin.
A distinct band of the size expected for Eurocin was seen in most
transformants whereas this band was not seen in the untransformed host
strains A. oryzae BECh2.

Example 4

Purification of Eurocin

[0275] The fermentation broth was filtered through a 0.22 μm Corning
431118 filter and pH adjusted to 4.0 using formic acid. Filtering through
a 0.22 μm filter was repeated to remove additional precipitate after
adjusting pH. The conductivity was measured and adjusted to approximately
10 mS cm-1 by adding MQ-water (Millipore). The filtrate was then
loaded onto a 12 ml Source 30S (Amersham Biosciences 17-1273-01) column
using an AKTA explorer HPLC instrument. Loading buffer was 50 mM formic
acid, pH 4 (buffer A).

[0277] Fractions containing the relevant compound (based on MALDI-TOF MS)
were pooled and concentrated on an Amicon Ultra 5000 Da MWCO filter
(Millipore UFC900524). Centrifugation was done at 4000 g for
approximately 30 minutes. The solution was then desalted by washing three
times with 0.01% acidic acid using the same filter and centrifugation
conditions.

[0278] The product was analyzed for correct sequence and purity by amino
acid analysis and MALDI-TOF MS. The measured mass of Eurocin was 4339 Da.
The theoretical molecular weight is 4338.8 Da, average, based on amino
acids 1 to 42 of SEQ ID NO:2.

[0279] The stock solution was stored at -20° C.

Example 5

Evaluation of Antimicrobial Activity

[0280] Purified Eurocin was evaluated for antimicrobial activity in a
microdillution assay. The

[0284] Sequence analysis using hidden markov model profiles (HMM profiles)
may be carried out either online on the Internet or locally on a computer
using the well-known HMMER freely available software package. The current
version is HMMER 2.3.2 from October 2003.

[0285] The HMM profiles may be obtained from the well-known PFAM database.
The current version is PFAM 16.0 from November 2004. Both HMMER and PFAM
are available for all computer platforms from e.g. Washington University
in St. Louis (USA), School of Medicine (http://pfam.wustl.edu and
http://hmmer.wustl.edu).

[0292] An amino acid sequence belongs to a PFAM family, according to the
present invention, if it generates an E-value which is greater than 0.1,
and a score which is larger or equal to zero, when the PFAM database is
used online, or when the hmmpfam program (from the HMMER software
package) is used locally.

[0293] When the sequence analysis is carried out locally using the hmmpfam
program, it is necessary to obtain (download) the HMM profiles from the
PFAM database. Two profiles exist for each family; xxx_ls.hmm for glocal
searches, and xxx_fs.hmm for local searches ("xxx" is the name of the
family). That makes a total of ten profiles for the five families
mentioned above.

[0294] These ten profiles may be used individually, or joined (appended)
into a single profile (using a text editor--the profiles are ASCII files)
that could be named e.g. defensin.hmm. A query amino acid sequence can
then be evaluated by using the following command line: [0295] hmmpfam-E
0.1 defensin.hmm sequence_file [0296] wherein "sequence_file" is a file
with the query amino acid sequence in any of the formats recognized by
the HMMER software package.

[0297] If the score is larger or equal to zero (0.0), and the E-value is
greater than 0.1, the query amino acid sequence is a defensin according
to the present invention.